Genetics - Theses
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ItemUsing insecticides to probe nicotinic acetylcholine receptors in Drosophila melanogaster( 2015)Insecticides remain the most effective means of insect control for both our personal protection and for the protection of our food and economic crops. The knowledge gained through the study of current and past insecticides can be a valuable tool in both the design of new, more effective insecticides and as a guide for integrated pest management. To best utilise a new insecticide and for it to retain field efficacy, its mode of action on its molecular target must be thoroughly understood. For this, a genetically tractable model organism can be used to enhance the understanding of insecticide:insect interactions through characterisation of resistance alleles, analysis of the insecticide target(s) and other resistance mechanisms. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. In this study, the vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. Recently an in vivo system was developed that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds. Rescue of the spinosad resistance phenotype in the Dα6 loss of function mutant, was possible with not only individual isoforms of this gene, but also with pest α6-like orthologues. It has also been found that a chimera of the Dα7 N-terminal and Dα6 C-terminal region is able to rescue the response to the insecticide spinosad. In this study, an incompletely dominant, spinosad resistance mechanism that may evolve in pest species is examined. First generated using chemical mutagenesis, the Dα6P146S mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system, the first reported use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair receptor gating. The Dα6 in vivo expression system was used here to assist in characterizing this dominant allele. The results of this study enhance our understanding of nAChR structure:function relationships, in particularly the interaction between spinosad and the Dα6 subunit. A complete in vivo rescue model was developed here for analysis of the Da1 subunit. A Dα1 deletion was generated using ends-out gene knockout technology and this knockout was found to be highly resistant to neonicotinoid insecticides. By combining this deletion with the GAL4:UAS binary expression system this study was able to rescue the phenotype of susceptibility to neonicotinoids as well as confirm the resistance potential of two nAChR subunits from the pest species, Helicoverpa armigera. This system is also used to investigate other phenotypes of the Dα1 deletion. The endogenous role of the nAChRs may enhance our understanding of why these are such effective insecticide targets, but also why they appear to be functionally redundant in terms of insect viability. Knowledge of other phenotypes present in lossof function mutants gives an insight into their function as well as hinting at fitness costs that may be associated with their loss. Targets with important roles are less likely to evolve resistance mutations if they also impact endogenous functions. If the target does have an important function, resistance modifications may lead to decreased fitness that would not be able to persist in natural populations without insecticide selection. A phenotype for the Da1 subunit was discovered whereby it appears to play a role in Drosophila courtship and mating behaviour. Although this allele can be cultivated in laboratory conditions, it would cause dramatic fitness effects under field conditions. Field evolved resistance mechanisms have been identified for both insecticides classes used in this study. Furthermore the neonicotinoid, imidacloprid, has recently been implicated in colony collapse disorder that is currently impacting honeybee populations. The results in this study provide information about the basic biology of the molecular targets of these insecticides. This information may help extend the life of these insecticides through more efficient use or perhaps provide ideas for new, more specific insecticides.
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ItemNicotinic acetylcholine receptors; an examination of expression and insecticide interactions in Drosophila melanogaster( 2012)Nicotinic acetylcholine receptors (nAChRs) are complex transmembrane proteins that belong to the Ligand-gated ion-channel (LGIC) super-family. They are responsible for cholinergic synaptic transmission in the central nervous system (CNS), a function conserved from worms to humans. The insect nAChR is a pentamer of α subunits, or a heteromer of α and β subunits and 10 subunits have been reported in Drosophila. Vast diversity is generated through different subunit assembly, RNA editing and alternative splicing. Thousands of subtle and noticeable pharmacological and electrophysiologically diverse receptors could be assembled. In insects, nAChR’s are targets of insecticides used to control pests. Chapter two describes work on the characterization of nAChR subunit genes in the central nervous system (CNS) of an embryo and larval D. melanogaster stages through in-situ hybridization, Fluorescent in-situ hybridization (FISH) and enhancer studies. Expression of 7 of the nAChR subunits (Dα1, Dα2, Dα3, Dα5, Dα6, Dα7 and Dβ2) was observed in CNS of embryo and larval CNS tissues. Beside the CNS, expression was also observed in other tissues, such as the ring glands (Dα1, Dα5, Dα7 and Dβ2) suggesting a role for these in the developmental biology of Drosophila. Salivary gland expression was observed for Dα7 subunit while larval fat body and adult hemolymph expression was observed for the Dβ3 subunit gene suggesting novel roles for these nAChR subunits. Building on the expression of these individual nAChR subunits, co-localization was also observed for Dα1/Dα2 and Dα1/Dβ2 subunit genes in larval CNS using FISH. In the third chapter a new approach was taken using RNAi as a tool for predicting insecticide resistance before it happens and finding new insecticide targets. Ten of the nAChR subunit genes were knocked-down using RNAi lines in the CNS of Drosophila. Results suggest that Dα6 is the only subunit targeted by the insecticide spinosad. Also individual knockdown of the Dα1 and Dβ3 subunits show significant sensitivity to spinosad, suggesting some form of compensation mechanism for these nAChR subunits. Conclusions from this work were that RNAi is an excellent tool in Drosophila (due to the availability of RNAi lines) in predicting resistance to insecticides, and prior testing of compounds could assist with better management of resistance development in insect pest species as insecticide targets are commonly conserved among insect species. The fourth chapter examines a negative cross-resistance of spinosad and nitenpyram resistant strains and by using mixtures of these insecticides to detect possible synergistic interactions. Negative cross-resistance was confirmed in earlier studies carried out by T. Perry (2005); a nitenpyram resistant mutant Dα1ems1 was observed to be sensitive to spinosad and a spinosad resistant mutant Dα6ems6 showed sensitivity to nitenpyram insecticide. My work using a number of mixture ratios found significant synergism between nitenpyram and spinosad insecticides at a ratio of 75 to 1. This synergistic ratio was found to be effective against the target site resistant mutants of nitenpyram and spinosad and also against a metabolic resistance mechanism to nitenpyram, indicating that mixtures can overcome both metabolic and target site resistances. My discussion chapter (Chapter 5) evaluates the expression studies and possible functions associated with the expression patterns observed in a particular tissues/life stage of Drosophila. It examines the advantages of some of our techniques such as RNAi as a fast method of predicting resistance. The implications of the negative cross-resistance relationship between spinosad and nitenpyram insecticides and the use of these two in mixtures are discussed with reference to resistance management and touches on future directions and ideas of practical implications of this in the field.